Increasing evidence supports the involvement of systemic inflammation in cancer development and progression (Mantovani et al., 2008). Apart from their well-established function as effector cells against invading pathogens, it is certain that neutrophils are involved in carcinogenesis (Hofman, 2010). Our current work focus on non-small cell lung cancers, because of the lack of predictive biomarkers and therapeutic targets. Moreover, for this cancer, metastatic diseases remain the foremost cause of cancer-related death.

Our project is divided into the following objectives:

i/ To characterize the role of the microenvironment on PMNL maturation (aims 1-2),

ii/ To decipher the involvement of PMNL and miRNA transfer in epithelial cell transformation (aims 2-3), and iii/ in tumour progression and dissemination (aim 4).

From a basic research point of view — Implementation of this project will help to define not only the sequence of events occurring from chronic inflammation to cancer onset, but also the specific fingerprint of PMNL recruited at sites of inflammation and/or within the tumour microenvironment.

Both in vitro, in vivo and ex vivo strategies will be used to accomplish our goal. We have already developed cellular and mouse models of colonic and lung cancer that allow us to test our approaches prior to clinical translation. Such an integrative strategy is expected to lead to much greater accuracy and cost-effectiveness than either strategy alone. Three scientists (two CR1 INSERM and one CR1 CNRS) together with post-doctoral fellows and doctoral students with a broad range of expertise in animal models, PMNL biology, miRNA/genotype and analysis of autophagy will dedicate 100 % of their time for this project.

The bench-to-bedside translation will be accomplished through the integration of the clinical translation core (7 physicians: three surgical pathologists, one pneumologist, one gastroenterologist, two surgeons, all of them strongly involved in translational and clinical research).

Taken together, we will answer the important question of how neutrophil/cancer cell crosstalk may impact on tumourigenesis and tumour progression and dissemination. Not only will our study bring fundamental knowledge but will also lead to the identification of new potential biomarkers and therapeutic targets of significant interest to the field of oncology.

Role of neutrophil-rich microenvironment in carcinogenesis onset and carcinoma progression

Several epidemiological and histological data highlight that intense and repeated neutrophil infiltration for a long period time is strongly linked with a high risk of carcinoma onset. However, eventhough different mechanisms were claimed to explain onset of carcinogenesis in response to repeated neutrophil transepithelial migration and/or neutrophil epithelial contact, the pathophysiological of this process is complex and its understanding is limited to date. Within a tumour, the cancer cells are surrounded by an inflammatory microenvironment containing different cell’s subtypes. Accumulating evidence highly suggests that cancer cells attract inflammatory cells, in particular polymorphonuclear leukocytes (PMNL, ie neutrophils) and might subvert their functions to promote tumour cell proliferation, resistance again cell death and metastasis. However, the relationship between cytokines, epithelial cells and PMNL, and the consequence of such cell-cell interaction on regulation of downstream events (such as miRNA regulation and/or protein expression), has been poorly investigated, in particular during the critical transition from a chronic “active” inflammatory lesion to a carcinoma.

Our project is divided in three main objectives : i) to characterize the role of microenvironment in PMNL maturation (aim 1), ii) to characterize PMNL involvement in tumor initiation (aim 2) and in tumor progression and dissemination (aim 3)

To conduct this project, we will use an integrative strategy which combined in vitro, in vivo and ex vivo studies. Our molecular and cellular approaches will be facilitated by different state-of-art facilities available in our pathology laboratory and our human biobank (www.biobank06.com).

From a basic research point of view — Implementation of this project will help to define not only the sequence of events occurring from a chronic inflammation to a cancer onset but also the specific fingerprints of PMNL recruited at site of inflammation or at tumour microenvironment.

From a clinical research point of view — Most of the current chemotherapies directly kill cancer cells; a strategy that however meets rapid drug resistance in large part due to the inherent genomic instability of cancer cells. We propose that tumour-associated PMNL (TANs), which are highly concentrated in tumour microenvironment, are promising biomarkers and potential targets for new drug development.

The broad translational potential of this project resides in its ability to provide validated targets for :

1) Assessing the genetic susceptibility of individuals to develop cancer,

2) Development of early prognostic biomarkers for management of cancer patients.

Aim 1 : Control of neutrophils maturation : Potential role of microenvironment

Strikingly, we frequently observe within human carcinomas, such as lung carcinoma, that intratumoral PMNL can be tightly associated with tumoral cells in areas without necrosis, but also around large areas of necrosis. This observation raises the following question: are there two populations of tumour-associated PMNL (TANs), one cytotoxic associated with dead tumor cells and one pro-tumoral in close proximity with alived tumor cells ?

In agreement with this proposal, Friedlander et al described in a mice model of lung cancer two TANs populations that are drived by TGFb signalling (Fridlender et al., 2009). Our general aim is to characterize the difference in PMNL subpopulation present respectively in a non tumoural and in a tumoural microenvironment.

Results based on tumoral microenvironment dependent subversion of PMNL functions will enable us to propose new mechanisms and biomarkers of tumour initiation.

Aim 2 : Role of PMNL in tumour initiation

Recent genome wide association projects highlighted single nucleotide polymorphisms (SNP) as cancer susceptibility genes in the general population (Stacey et al., 2009). However, most of these SNP are silent and therefore their pathological consequences were not studied to date. We recently demonstrated that an inflammatory environment reveals SNP’s pathological consequences via disturbing miRNA-mRNA binding (Brest et al., 2010b).

Interestingly numerous genetic variations are present on genes involved genome stability. We therefore will focus our attention on cancer-associated SNP that could affect genome stability. The core of our proposal lies on the hypothesis that even minute decrease in DNA stability/repair may significantly increase the risk of developing cancer over lifetime, particularly in response to PMNL-rich microenvironment.

Our general aim is to investigate if a PMNL rich-microenvironment might promote cancer development by disturbing genome stability.

We believe that results of this study that may support a role for SNP/miRNA regulation of telomerase and telomere stability will enable us to improve individualized risk prognosis and could assist in the development of personalized new therapeutic strategies.

Aim 3 : Role of TANs in circulating tumor cells dissemination

Metastasis is the leading cause of death in cancer patients. This is likely because the mechanisms behind tumour dissemination, through circulating tumor cells (CTC) present in the blood stream are not fully understood

Using a state-of-art platform for detecting CTC by using both indirect and direct approaches, we recently proposed CTCs as valuable index of the risk for developing metastases in lung carcinoma (Hofman et al., 2010a; Hofman et al., 2010b). Moreover, we made the original observation that PMNL frequently stick to CTC clumps which raises the question: are CTC co-migrate with PMNL ?

Our general goal is to study CTC biology, particularly their dialogue with TANs

We believe that results of this study will enable us to assist in the development of early biomarkers of tumor dissemination.

RELEVANCE FOR DIAGNOSTIC, PRONOSTIC AND THERANOSTIC

The achievement of this program benefits of

* the range of expertises possessed by the applicant team;

* the insertion of our team within the INSERM Biobank and Laboratory of Clinical and Experimental Pathology (www.biobank06.com) ;

* the development of different in vitro, in vivo and ex vivo tools; and

* national and international collaborations

* Such unique synergy should greatly speed up the translation of our findings into the clinical practice.

HEBUTERNE Xavier, PH-PU1E-mail :
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HOFMAN Véronique, PHE-mail :
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ILIE Marius, AHUTel. +33 (0)4 92 03 16 02, E-mail :
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MARQUETTE Charles-Hugo, PU-PHE-mail :
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MEYER Mickaël, MENRT Thesis

MOGRABI Baharia, CR1 INSERMTel. +33 (0)4 92 03 12 43, E-mail :
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MOUROUX Jérôme, PU-PH1E-mail :
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MUSSO-LASSALLE Sandra, MCU-PH2E-mail :
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